Gel Filtration Chromatography — Creative Biostructure
Gel Filtration Chromatography
MagHelix™ Protein Purification platform has been established to perform high-quality recombinant protein purification based on our advanced Protein Production Service. Creative Biostructure is not only the world leading protein manufacturer, but also the expert to provide protein purification technical support in order to accelerate your research.
Gel filtration, as known as size exclusion chromatography (SEC), separates proteins according to their different size as they pass through a gel filtration column. Compared to affinity chromatography or ion exchange chromatography, proteins to be seperated by gel filtration chromatography do not need to bind to the chromatography medium, making buffer composition hardly affect resolution. The major advantages and disadvantages of gel filtration chromatography are listed as below:
There are three main applications of gel filtration chromatography:
1. In group separations
Adjusting pH, buffer type, salt concentration during sample preparation;
Removing interfering small molecules (EDTA, Gu-HCl, etc);
Removing small reagent molecules (fluorescent labels, radioactive markers, etc);
But not when the protein will precipitate.
2. In fractionation
Excellent during the polishing stage;
Removes dimers and other aggregates;
Transfers protein to buffer solution ready for the next set of experiments;
Not so suitable if the sample volume is large.
3. For size estimation
Gives an estimate of molecular size in any practically any solution;
Precision is not so good.
1. Poor resolution
Reason: Poor packed bed
Recommended solution: Check the efficiency of the column and re-pack if necessary.
Always check the efficiency of a new column, especially one that has been packed in the lab, before using it for critical applications.
Reason: Contamination of the bed with lipids, denatured protein etc.
Recommended solution: Thoroughly clean the column following the recommended protocols given in the instructions provided by the column manufacturer.
2. Poor yields
Reason: Contamination of the bed with lipids, denatured protein etc.
Recommended solution: Thoroughly clean the column following the recommended protocols.
Tip: Running eluent slowly through the column can prevent microbial growth. Cleaning the column thoroughly and keep it under cold conditions in case that the column will not be used for a long time.
3. Elution earlier than expected
Reason: Ionic aggregates and complexes due to carbohydrate chains (glycoproteins).
Recommended solution: Include 150mM NaCl or up to 10% betaine in buffer.
Reason: Complexes associated via SH-groups.
Recommended solution: Add reducing agent (e.g. DTT) to buffer.
Reason: Hydrophobic aggregates.
Recommended solution: Add 10-20% ethanol or other organic solvents to buffer.
Reason: Gel filtration of micelles/protein aggregates.
Recommended solution: Decrease buffer detergent concentration until below CMC or use a different detergent (stay below its CMC).
Remember to check that your sample and your column are stable in the buffer before using special additives to aid solubilisation.
4. Elution later than expected
Reason: Digestion by proteases.
Recommended Solution: Add protease inhibitors to buffer.
Reason: Hydrophobic adsorption.
Recommended Solution: Add 10-20% ethanol or other organic solvents to buffer or decrease salt concentration in high ionic strength buffer.
Reason: Ionic adsorption due to negatively charged groups on the support (Carboxyl-, Sulphate-) and positively charged roups on sample molecules.
Recommended Solution: Include 150mM NaCl (or other salt) in buffer.
TIP: Be sure that your sample is stable in the buffer additive prior to use. Some additives recommended here may denature or otherwise inactivate a protein.
TIP: Be sure your column and system are stable in the buffer additive prior to use.
Creative Biostructure has a professional support team to provide a wide variety of technical resources in protein purification that are listed as below. We are happy to help you and address your research problems. Please feel free to Contact Us.
Applications of Electrophoresis
Electrophoresis is a widely used technique in molecular biology. Using an electrical field, molecules are separated, usually by size, in a gel matrix. Electrophoresis can be used on a huge range of biological molecules, but is more prominently used with DNA and Proteins. Our electrophoresis gel tanks consist of 3 main components:
- A buffer chamber, to hold the gel conductive buffer
- A gel support, either a tray, for horizontal systems, or glass plates, for vertical systems
- Electrodes to transmit the electrical field through the buffer
There are slight differences in the electrophoresis tank designs used for DNA and Protein, and each uses a different type of gel.
DNA Electrophoresis in Agarose Gel
DNA is negatively charged due to the phosphate groups of the structural backbone of the molecule. This means that when DNA is exposed to an electrical field in conductive buffer, it will move towards the positive electrode of the system. Without any resistance, this would lead to a rapid and disorganized flow of DNA through the buffer. To organise the movement of the DNA, we introduce a separation matrix, made of a sugar called agarose. By melting agarose powder, dissolving it in conductive buffer and allowing it to set, a semi-solid gel is formed. This gel contains microscopic pores, present due to the loose cross-linking of the agarose. The pores block the migration of the DNA, slowing down large molecules more than smaller ones. When the DNA is visualised using fluorescent or visible stains, this resistance results in a distinctive pattern of bands associated with DNA of different lengths.
Our Agarose gel tanks are suitable for use in routine agarose gel electrophoresis, and come in a range of sizes for different sample number and resolution lengths.
Advantages and application of the Raptor gel
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Application software is responsible for the efficient functionality of computing devices. The advantages and disadvantages of application software are enlisted in the article below.
Our computers and mobile devices are all loaded with all kinds of software, and each software has a different purpose to fulfill. It is important to first understand the differences between application software and system software. System software is the programming and the coding that is utilized by the various components in the device to communicate with each other.
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There are millions of commands and instructions that are transferred from one component to another with every passing second, and the system software enables this flow of communication. Additionally, the person using the device does not directly interact with or use the system software, since it is all happening inside the device itself.
On the other hand, application software is that which is used directly by the user for the sole purpose of completing a certain task. This type of software needs to be installed separately on a device, and it regularly has interfaces that enables communication between the machine and the user, hence giving rise to many different advantages and disadvantages.
For instance, consider a program that allows you to work with databases. This is a software that is used for a specific application, so it falls under this category. It is universally assumed that they have many advantages, and you will be hard pressed to find disadvantages for the same because ultimately, the purpose of all these devices is to perform many tasks with their help.
Common Types of Application Software
The different types of application software are used by individual users and business enterprises as well, and they have many benefits of doing so. This includes word processing software, database software, multimedia software, editing software, and many other different kinds as well. All these software are either provided individually, or they are packaged together and sold by business to business sellers. When a whole variety of them are integrated collectively and sold to a business, they can take up the form of enterprise software, educational software, simulation software, information worker software, etc.
When you begin to compare, you will find that the pros outweigh the cons very easily. With that in mind, here are some of their most popular and widely accepted benefits. Note that in this scenario, we are speaking of application software that is designed for a specific purpose, to be used either by individuals or by businesses.
- Their single biggest advantage is that they meet the exact needs of the user. Since they are designed specifically with one purpose in mind, the user knows that he has to use one specific software to accomplish his task.
- The threat of viruses invading custom-made applications is very small, since any business that incorporates it can restrict access and can come up with means to protect their network as well.
- Licensed application software gets regular updates from the developer for security reasons. Additionally, the developer also regularly sends personnel to correct any problems that may arise from time to time.
As is the case with all such matters, there are certain disadvantages of such software as well. Though these are not spoken about very often, nor are they highlighted, the fact is that they do exist and affect certain users. But people have accepted these misgivings and still continue to use such software because their utility and importance is much more profound than their weaknesses.
- Developing application software designed to meet specific purposes can prove to be quite costly for developers. This can affect their budget and their revenue flow, especially if too much time is spent developing a software that is not generally acceptable.
- Some software that are designed specifically for a certain business, may not be compatible with other general software. This is something that can prove to be a major stumbling block for many corporations.
- Developing them is something that takes a lot of time, because it needs constant communication between the developer and the customer. This delays the entire production process, which can prove to be harmful in some cases.
- Application software that is used commonly by many people, and then shared online, carries a very real threat of infection by a computer virus or other malicious programs.
So, whether you are buying them off the shelf, or whether you are hiring a developer to build specific software for you, all of these points will seem pertinent to you. Many individuals and businesses have regularly found the need and the requirement for such software, and the fact remains that any computing device will be utterly useless without such software running on it.
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There are many types of computer software, like testware, firmware, system software, middleware, etc. The article below gives you some examples of application software.
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